1. Field of the Invention
This invention is concerned with the phenomenon of hyperaccumulation of metals in plants and the removal of metals from contaminated substrates by plants. The invention relates in particular to methods for removing metals from soil or other environments using modified plants having an increased capacity for metal uptake, and to methods for obtaining such plants. The invention is also concerned with transgenic plants having an increased capacity for metal uptake, and to recombinant vectors and transformed plant cells used to obtain the transgenic plants.
2. Description of the Related Art
Many soils are contaminated by metals that derive from sources such as mine workings, metal-processing industries, atmospheric depositions, the power and fuel industries, deposition of contaminated sewage sludge, and others. These contaminated substrates pose serious human health problems, are deleterious to the environment, and can render crop cultivation either inadvisable (because of the health hazard from the plant products) or impractical (because of the toxicity of the metals towards plants). These problems are becoming more acute in many regions, for example as a result of increased deposition of contaminated sewage sludge on land rather than at sea.
Conventional approaches for decontaminating metal-polluted soils have relied on physical methods such as landfilling (the excavation and removal of soil to a landfill site designated for hazardous waste), fixation or immobilization (such as infiltration with cement or by vitrification), or leaching (for example with strong acids to desorb the metal ions) (see Salt, D. E. et al. (1995) Bio/Technology 13, 468-474). These methods are not only expensive but are environmentally less than ideal, for example because they tend to destroy soil structure (as well as the microbial organisms in the soil), and can give rise to secondary pollution in run-off water. Thus it has been recognized that removal of metals from contaminated soils by metal-tolerant plants might provide an alternative and cost-effective technology for environmental clean-up, a concept known as xe2x80x9cphytoremediationxe2x80x9d (Chaney, R. L. (1983) In Land Treatment of Hazardous Wastes, eds Parr, J. F., Marsh, P. B. and Kla, J. M., pp 50-76. Noyes Data Corp., Park Ridge, N.J.; Salt et al. (1995), loc. cit.).
To date, the application of plants in phytoremediation has been limited by the severe toxicity of most metal ions to the majority of plant species. However, a number of plant species are known that are both relatively metal tolerant and capable of accumulating metals to high concentrations in their above-ground biomass. These plants are known as xe2x80x9cmetal hyperaccumulatorsxe2x80x9d, of which several hundred species have so far been described (Baker, A. J. M. and Brooks, R. R. (1989) Biorecovery 1, 81-126). These plants have favourable characteristics for bioremediation, since the above-ground parts (leaves and stems) can be readily harvested, and the metal-rich residues processed in a controlled manner. Successive cycles of crops of these metal-hyperaccumulator plants would be expected to lead to progressive decontamination of the soils, and this has been demonstrated (Baker, A. J. M. et al. (1994) Resources, Conservation and Recycling 11, 41-49; Brown, S. L. et al. (1994) J. Environ. Qual. 23, 1151-1157; Brown S. L. et al. (1995) Soil Sci. Soc. Am. J. 59, 125-133). However, most hyperaccumulator plants are of relatively small stature and are slow-growing, so long periods of time would be needed to decontaminate most metal-polluted substrates to acceptable levels (Baker, A. J. M. et al. (1994) loc. cit.). The definition of xe2x80x9cmetal hyperaccumulatorxe2x80x9d varies according to the metal concerned, e.g. for zinc it is  greater than 1% by dry weight, while for nickel and cobalt it is  greater than 0.1% by dry weight (see Baker, A. J. M. et al. (1994) loc. cit.).
The underlying mechanism of metal-hyperaccumulation in plants has not up to now been understood. Most theories of metal tolerance in plants have assumed that metal ions within the plant are detoxified by chelation with an appropriate high-affinity ligand (Ernst, W. H. O. et al. (1992) Acta Bot. Neerl. 41, 229-248). The most frequently suggested ligands of this type have been cysteine-rich proteins called metallothioneins (Robinson, N. J. et al. (1993) Biochem. J. 295, 1-10), including the lower-molecular-weight phytochelatins (Rauser, W. E. (1990) Annu. Rev. Biochem. 59, 61-86), and organic-acid anions such as malate, citrate and malonate (Reeves, R. D. (1992) In The Vegetation of Ultramafic (Serpentine) Soils, eds Baker, A. J. M., Proctor, J. and Reeves, R. D., pp 253-277. Intercept Press, Andover). However, the concentrations of these potential metal-chelating ligands do not respond in the proportional and metal-specific manner that would be anticipated if they had a fundamental role in the phenomenon of metal hyperaccumulation (Ernst, W. H. O. et al. (1992) loc. cit.; Reeves, R. D. (1992) loc. cit.; de Knecht, J. A. et al.(1994) Plant Physiol. 104, 255-261).
In principle, if the biochemical mechanisms responsible for metal tolerance and metal accumulation in plants were understood, this would permit the development of novel strategies for the application of plants in the clean-up of contaminated soils. For example, genetically modified plants with altered biochemical characteristics can be generated using recombinant DNA techniques (xe2x80x9cgenetic engineeringxe2x80x9d). Several reports have been published of plants genetically transformed to express one or other animal metallothionein gene, but these have given variable results. In some (but not all) experiments, an increase in tolerance towards cadmium was observed in plants expressing an animal metallothionein gene, but this appeared to be associated with a decreased cadmium accumulation in the above-ground parts of the plant (Elmayan, T. and Tepfer, M. (1994) Plant J. 6, 433-440, and references therein). Plants genetically modified in this manner thus do not appear to be suitable for the purpose of phytoremediation of contaminated soils.
Novel strategies for the application of plants in the extraction of metals from soils would be possible if the biochemical processes responsible for the phenomenon of metal hyperaccumulation were understood. The so-called xe2x80x9cmetal hyperaccumulator plantsxe2x80x9d have the ability to extract metals effectively from the soil (cf. Bernal, M. P. et al. (1994) Plant Soil 164, 251-259), to accumulate high amounts of metals in their above-ground biomass, and to tolerate metal concentrations in the soil that would be toxic to the great majority of plant species (Baker, A. J. M. and Brooks, R. R. (1989) loc. cit.). These properties are all highly desirable in plants to be used for purposes of phytoremediation. Up to now, however, there has been no clear understanding of the cellular factors responsible for these distinctive features of metal hyperaccumulator plants.
Recently, it has been observed that the amino acid histidine increases in the xylem sap of the hyperaccumulator plant Alyssum lesbiacum when these plants are exposed to nickel in the root medium (Kramer, U., Baker, A. J. M. and Smith, J. A. C. (1994) Abstracts of the Fifth International Symposium on the Genetics and Molecular Biology of Plant Nutrition, University of California, U.S.A., July 17-24; Smith, J. A. C., Kramer, U. and Baker, A. J. M. (1995) Meeting on Phytoremediation by Hyperaccumulator Plantsxe2x80x94Current Research and Future Requirements, Rothamsted, UK, January 25-28; Smith, J. A. C., Kramer, U., Tibbetts, R. A. and Baker, A. J. M. (1995) Second International Conference on Serpentive Ecology, Noumxc3xa9a, New Caledonia, July 31xe2x88x92August 5). When supplied to the xylem of excised shoots of the non-metal-accumulating species A. montanum, histidine apparently reduced the toxicity of nickel; also, when apparently supplied to the root medium of excised roots of this species, histidine again apparently reduced the toxicity of nickel, as manifested by an increased exudation of sap from the cut xylem and increased flux of nickel through the root system (Smith, J. A. C., Krxc3xa4mer, U. and Baker, A. J. M. (1995) Fourteenth Annual Symposium, Current Topics in Plant Biochemistry, Physiology and Molecular Biology, University of Missouri, Columbia, Mo.,
The present invention aims to overcome the problems associated with conventional soil decontamination methods, and to provide methods and means for decontamination of metal-polluted soils and other plant-supporting environments using phytoremediation technology.
The inventors have now discovered that the production of histidine in response to metal exposure is functionally responsible for both the high degree of metal tolerance shown by so-called xe2x80x9cmetal-hyperaccumulator plantsxe2x80x9d in the genus Alyssum and their characteristic ability to accumulate very high concentrations of metals in their shoots. We report here that the nickel accumulated within the plant is chelated with histidine. We have also demonstrated that surprisingly, by simple application of histidine as a foliar spray, the nickel tolerance of a normally non-nickel-tolerant species of Alyssum is greatly increased. We have also recognised that, given the association of the hyperaccumulation trait with a specific metabolic pathway, the transfer of a particular gene involved in that metabolic pathway to a target plant species can be used to give transgenic plants which both accumulate high concentrations of metals and show favourable growth characteristics for phytoremediation.
The present invention therefore provides in one aspect a method of removing an amount of metal from a metal-containing substrate, which method comprises:
a) identifying a metal-containing substrate;
b) providing modified plants having an improved metal accumulating capability compared to the unmodified plants by virtue of an increase in the concentration of histidine within the modified plants; and
c) maintaining at least one modified plant on the metal-containing substrate under conditions such that the modified plant accumulates the metal from the substrate.
The metal-containing substrate may be a soil environment or other environment capable of supporting plant growth, and could be a purely aqueous environment.
In a preferred embodiment of the method according to the invention, the modified plants are genetically engineered plants modified so as to be capable of increased production of histidine compared to their non-modified counterparts.
In another embodiment, the increased concentration of histidine within the plants is achieved by administering a formulation comprising an effective concentration of histidine to the plants. The formulation may be conveniently administered as a foliar spray. The foliar spray would need to contain a relatively high concentration of histidine and be administered at regular intervals. By this method, histidine enters the plants either through the foliage in particular the shoot surfaces, or via the root system, or via both routes.
In another aspect, the invention provides a method of improving the metal accumulating capability of a selected plant, which method comprises transforming plants cells of the selected plant with a recombinant vector comprising a nucleic acid sequence which encodes a polypeptide capable of augmenting the concentration of histidine in the plant cells, the nucleic acid sequence operably linked to an expression control system capable of effecting the expression of the nucleic acid sequence in the plant cells. Successful transformants are selected for and used to produce regenerated transgenic plants.
In further aspects, the invention provides recombinant vectors capable of transforming plant cells in the manner described above, plant cells or plant cell cultures transformed by the vectors, methods of obtaining the vectors and transformed cells, and regenerated transgenic plants having an improved metal accumulating capability compared with their non-transgenic counterparts.
The improved metal accumulating capability achieved according to the invention involves an increase in metal tolerance and/or rate of metal uptake, preferably both. Plants which are not already metal hyperaccumulators may thus be rendered metal hyperaccumulators, although this is not a requirement of the invention. The desired objective is to achieve an increase in the rate of metal extraction out of a unit quantity of soil or other substrate.
The metal can include as well as nickel, cobalt, or zinc, such elements as aluminium, americium, antimony, arsenic, barium, beryllium, bismuth, cadmium, caesium, cerium, chromium, copper, gallium, germanium, gold, indium, iridium, iron, lead, manganese, mercury, molybdenum, neptunium, osmium, palladium, platinum, plutonium, radium, rhenium, rhodium, rubidium, ruthenium, scandium, selenium, silver, strontium, technetium, tellurium, thallium, tin, tungsten, uranium, vanadium, yttrium, including stable or radioactive isotopes of the above. Also included are metalloids, one or more metals in combination, and metals associated with any or various combinations of organic compounds such as oils, fats, grease, fuels, kerosene, phenols, benzene or detergents. The accumulation by plants of any soil contaminants is of interest. In plants having improved metal accumulation properties, elevated metal accumulation may be expected to extend beyond the metal usually bound by the histidine acting as a metal-chelating ligand. It will be a simple matter to test whether a particular modified plant according to the invention is capable of accumulating a significant amount of a particular pollutant in a soil environment.
The plants and plant cells derived from them for use in the invention are preferably chosen from the family Brassicaceae, which is expected to provide the most effective and practical species for this purpose. However, plants from other families are not excluded. Desirable features of plants for use in the invention are favourable growth characteristics for phytoremediation purposes, such as relatively fast growth rates, large and/or deep root systems beneficial for metal extraction from soil, and easy harvesting. Certain members of the genus Brassica are suitable in one or more of these respects.
The amino acid histidine is produced in all plants to some degree and also in microorganisms including bacteria and fungi, but not in mammals (for which it is one of the 10 or so essential amino acids). Thus, recombinant DNA technology can be employed to up-regulate histidine production in a selected plant, e.g. by xe2x80x9cover-expressingxe2x80x9d one or more of the key genes involved in the histidine biosynthetic pathway. Such over-expression may be achieved by up-regulating the expression of one or more of the plant""s own genes; or, more conveniently, it may be achieved by introducing one or more heterologous genes encoding a polypeptide or polypeptides active in the histidine biosynthetic pathway of the selected plant. Suitable heterologous genes may come from known metal hyperaccumulators such as certain species of Alyssum or from bacteria such as Escherichia coli. Advantageously, the plant into which they are introduced will have favourable growth characteristics e.g. as discussed above.
Techniques which may be used in the context of the invention for transforming plant cells and regenerating plants from the transformed cells are well-known to those skilled in the art and are reviewed in Gene Transfer to Plants (1995) eds. I Potrykus and G. Spangenberg, Springer-Verlag, Berlin. Transformation may be carried out using an Agrobacterium-based transformation system, with e.g. A. tumefaciens, or A. rhizogenes which specifically transforms roots. Alternative transformation systems include e.g. biolistic systems.
Techniques and components for construction of vectors according to the invention are also available to those skilled in the art. A vector needs to be chosen which is capable of transforming plant cells, more specifically higher plant cells and the cells of the chosen target or host plant which is to be modified. There will need to be included in the vectors along with the genetic material for increasing histidine production, appropriate expression control sequences which are functional in the selected plant to be transformed. These will usually include a suitable promoter, which may be a constitutively acting or a conditional promoter and could be organ-specific if desired. Preferably an appropriate transcription termination signal will also be present. Another feature which may be included is a signal for directing the polypeptide encoded by the vector to the plastid in a target plant cell, since histidine biosynthesis occurs mainly or exclusively in the plastid. Such a signal may be provided for example by a transit peptide as will be described in more detail later on.
It will be understood that plants having additional modifications concerned with characteristics other than histidine production are not excluded from the invention. In addition to having improved metal accumulation properties, the plants may have modifications, genetic or otherwise, that increase their suitability for phytoremediation.
The invention thus provides a strategy to produce plants with increased effectiveness over conventional varieties in phytoremediation, with an improved ability to accumulate nickel and/or other metals, by virtue of a genetically engineered increase in the amount of histidine within the plant, this property being expressed either constitutively or in response to exposure to metal.